Medium and method for detecting/identifying microorganisms

ABSTRACT

A medium for biological analysis by biochemical means involving chromogenic or fluorogenic substrates that react with enzymes (esterases and/or osidases and/or peptidases and/or sulfatases and/or phosphatases) specific for the target strains is that is in a stable, ready-to-use liquid or gel form and contains at least one emulsifying stabilizer of the fatty acid sorbitan ester/fatty acid type and at least one solvent (dimethyl sulfoxide). This medium is obtained by mixing a stock solution of esterase and/or osidase and/or peptidase and/or sulfatase and/or phosphatase substrates in the solvent, and the emulsifying stabilizer, with some of the constituents of the culture medium.

This application is a National Stage application filed under Rule 371from PCT/FR01/03611 filed Nov. 16, 2001 which claims priority fromFrance 00/14879 filed Nov. 17, 2000.

The field of the invention is that of microbiological analysis bybiochemical means and in particular the detection and identification ofbacterial strains by the inoculation of reaction media, particularlynutrient media. The latter comprise chromogenic or fluorogenicsubstrates that are capable of reacting with enzymes specific for thetarget strains to produce a coloration or a fluorescence for each colonyin question.

Of more particular interest within the framework of the presentinvention is the detection/identification of pathogenic orquality-indicating microorganisms, whether in a medical or industrialenvironment, and more particularly microorganisms with an enzymaticactivity of the esterase, osidase, peptidase, sulfatase or phosphatasetype, for example bacteria or yeasts of the genera Salmonella,Pseudomonas, Listeria, Staphylococcus, Enterococcus and Candida and moreparticularly those of the species Escherichia coli, Listeriamonocytogenes, Staphylococcus aureus and Candida albicans.

The presence of strains of Escherichia coli is often demonstrated byrevealing an enzymatic activity of the osidase type, such asβ-glucuronidase or β-galactosidase activity.

Likewise, the genus Listeria is detected by revealing the presence ofβ-glucosidase activity.

An aminopeptidase activity can also be utilized to reveal a group, agenus or a species of microorganisms. Alanine aminopeptidase activity,for example, makes it possible to differentiate between Gram-negativebacteria and Gram-positive bacteria.

The genus Salmonella, which is responsible for a variety of infections(typhoid fever, food poisoning) in humans, possesses non-specificesterases capable of hydrolyzing chromogenic, for example indigogenic,synthetic substrates.

The detection/identification of salmonellae, and more generally ofbacteria with esterase activity, is conventionally carried out on geloseor liquid isolating media that allow the detection/identification ofsuspect colonies of bacteria with esterase activity, especiallysalmonellae. The inoculation of such media is effected by immersing saidmedium in the sample analyzed or by bringing the sample into contactwith the medium.

In their enzymatic makeup, bacteria with esterase, osidase, peptidase,sulfatase or phosphatase activities possess esterases, osidases,peptidases, sulfatases or phosphatases which cleave the target linkagesof the substrates present in the medium and thus release the activatedchromophoric or fluorophoric part of said substrates. This results in acoloration or a fluorescence that reveals hydrolysis and hence thepresence of target bacteria or colonies of target bacteria.

To be able to perform routine tests on a large scale, it is necessaryfor the detection/identification media to be stable and to enable thecorresponding detection/identification methods to be simplified as faras possible by limiting the manipulations. Also, it is important for themethods to offer a high sensitivity (high intensity of coloration) and afirst order specificity. Another fundamental parameter of these types ofmedia and methods for the detection/identification of bacteriaexhibiting the above-mentioned enzymatic activities is the rate at whichthe suspect colonies are revealed.

Now, it is known that substrates of enzyme such as esterases, osidases,peptidases, sulfatases or phosphatases present problems of compatibilitywith culture media for microorganisms, particularly bacteria, possessingthese activities. Furthermore, such substrates are not stable over time,meaning that the sensitivity towards the enzymatic activity in questiondecreases with storage time.

In this context the scientific article entitled “Synthèse de substratsindigogéniques. Mise en évidence de l'activité estérasique dessalmonelles” (“Synthesis of indigogenic substrates. Demonstration of theesterase activity of salmonellae”): A. Agban et al., Eur. J. Med. Chem.(1990) 25, 697–699, discloses gelose culture media comprisingindigogenic substrates, namely 5-bromoindoxyl pelargonate (C9) inparticular, and a bile salt, namely sodium deoxycholate. Such culturemedia suffer from the same disadvantages as those referred to below withreference to patent document FR 2 697 028.

Patent FR 2 697 028 discloses a culture medium for revealing thepresence of salmonellae which comprises a chromogenic esterase substrateconsisting of an ester of caprylic acid with an indole radical(5-bromo-4-chloro-3-indolyl caprylate), together with a detergentselected from bile salts (sodium deoxycholate). This chromogen and thisbile salt are contained in a nutrient medium that allows salmonellae togrow. According to the teaching of FR 2 697 028, the bile salt is addeddirectly to the selective medium already containing the esterasesubstrate.

Another disadvantage associated with the use of bile salts derives fromthe fact that they are starting materials of animal origin, which varyin quality.

In addition, the results in terms of biological activity are capable ofimprovement.

This culture medium does not offer all the desirable guarantees in termsof stability of the esterase substrate. Moreover, the latter proves tobe incompletely miscible with the culture medium, which obviouslydetracts from the quality of the results obtained from the point of viewof sensitivity, rapidity and stability.

It must also be noted that the culture medium according to FR 2 697 028is in the form of a powder. This means that the user first has to carryout an operation to reconstitute the liquid or gelled medium. Thisconstraint is a consequence of the lack of stability of the esterasesubstrates used. In addition, the gelose medium prepared according tothe teaching of FR 2 697 028 is not translucent, which is likely tocompromise the reading of the colorations associated with any coloniesof target bacteria.

PCT patent application WO-92/17607 relates to a detection medium forsalmonellae which contains TERGITOL-4® (7-ethyl-2-methyl-4-undecanoatehydrogensulfate or its sodium salt). This additive is supposed toimprove the selectivity of the detection medium towards targetsalmonellae. The concentration of TERGITOL-4® can vary from 2 to 30 ml/lin the culture medium based on agar gelose/xylose/lysine. According tosaid document, the detection of salmonellae is based solely on aprinciple of selective growth by competition.

PCT patent application WO-99/41409 relates to chromogenic esterasesubstrates for the detection of salmonellae.

Said document proposes the use of a chromogenic compound which reactswith an esterase/lipase specific for the genus Salmonella and having anaffinity for C8 fatty acid esters. The chromogenic compound in questioncomprises an anion and a cation of the formula[4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)vinyl]-quinolinium-1-(propan-3-yl-)carboxylate]+,-[bromideor chloride]. More precisely, the substrates used are C8 esters, forexample, of the carboxylate cation.

Also, the method according to WO-99/41409 describes the use of asorbitan fatty acid ester (particularly the monolauric acid ester TWEEN®20). These products are employed as detergents at a rate of 2 g perliter of medium.

In addition, the specific substrate according to the teaching of saidPCT patent application can be introduced into the nutrient culturemedium as a mixture with methanol, ethanol or N,N-dimethylformamide(DMF). The detergent, if used, is introduced separately rather thantogether with the substrate and the solvent.

Whatever the case may be, the means disclosed in WO-99/41409 are notpresented as providing an improvement in the stability of the enzymesubstrates contained in the detection medium.

Various documents also disclose culture media containing severalsubstrates for the detection of a single enzymatic activity, as well asculture media containing several substrates for the detection ofdifferent enzymatic activities.

Patent document WO-95/04157, for example, describes culture mediacontaining different chromogenic osidase substrates such as5-bromo-4-chloro-3-indoxylgalactoside,5-bromo-4-chloro-3-indoxylglucuronide,5-bromo-4-chloro-3-indoxylglucoside or 6-chloro-3-indoxylgalactoside.Such media are used to differentiate especially between bacteria of thespecies Escherichia coli and other coliform bacteria.

Said document further describes a medium containing chromogenicsubstrates specific for different enzymes such as osidases andphosphatases. Examples of these substrates are5-bromo-4-chloro-3-indoxyl N-acetyl-glucosaminide and5-bromo-6-chloro-3-indoxyl phosphate. Such a medium makes it possible inparticular to differentiate between Candida albicans and other yeasts ofthe genus Candida.

Patent document WO-00/53799 describes a chromogenic medium for thedetection of Staphylococcus aureus bacteria. This medium contains twochromogenic substrates in combination, especially5-bromo-4-chloro-3-indo-xyl-glucoside and 5-bromo-6-chloro-3-indoxylphosphate. The principal purpose of such a medium is avoiding falsepositives or false negatives and differentiating between S. aureus andother species or other genera such as Streptococcus.

However, patent documents WO-95/04157 and WO-00/53799 give no indicationthat the media described were developed in order to improve thestability of the enzyme substrates they contain.

Fatty acid sorbitan esters (FASE) are known surfactants that are widelyused especially in food and pharmaceutical preparations. By way ofillustration, there may be mentioned the article by DICKINSON et al. in“J. Colloid Interface Sci. 1999 Apr. 15; 212 (2): 466–473”, whichrelates to the stabilization of emulsions containing sodium caseinateand a polyethoxylated sorbitan monolaurate containing 20 units ofethylene oxide (TWEEN 20). The emulsions in question are oil-in-wateremulsions (30% by volume of n-tetradecane at pH 6.8). This technicalfield is relatively remote from that of the detection of bacteria withspecific enzymatic activity by means of a color reaction on a selectiveculture medium.

The article by GRAM et al. in “Clin. Chem. 1985 Oct.; 31 (10): 1683–8”concerns the use of polyethoxylated sorbitan monooleate (TWEEN 80) in anantithrombin-III assay medium, said medium also containing polyethyleneglycol, a solution of thrombin and synthetic chromogenic peptidesubstrates. The additives used are presented as allowing an increase inthe concentration of active thrombin so as to guarantee that theanalysis performs well. Once again, one is forced to observe thattechnical concerns of this kind are very different from those peculiarto a medium for the detection of bacteria with specific enzymaticactivity using selective culture media containing chromogenicsubstrates.

In such a technical environment, one of the essential objectives of thepresent invention is to provide a medium for thedetection/identification of bacteria with an enzymatic activity selectedfrom esterase and/or osidase and/or peptidase and/or sulfatase and/orphosphatase activities which is made up in such a way as to optimize thesensitivity of the analysis, i.e. maximize the intensity of thecoloration or fluorescence revealing the presence of target bacteria.

Another essential objective of the present invention is to provide amedium for the detection/identification of microorganisms, especiallybacteria or yeasts, with an enzymatic activity selected from esteraseand/or osidase and/or peptidase and/or sulfatase and/or phosphataseactivities which is perfectly translucent before inoculation with thesample to be analyzed.

Another essential objective of the invention is to provide a medium forthe detection/identification of microorganisms, especially bacteria oryeasts, with an enzymatic activity selected from esterase and/or osidaseand/or peptidase and/or sulfatase and/or phosphatase activities whichcontains at least one chromogenic or fluorogenic enzyme substrateselected from esterase substrates and/or osidase substrates and/orpeptidase substrates and/or sulfatase substrates and/or phosphatasesubstrates and is storage stable (intensity of the revealed colorationor fluorescence maintained at a maximum level for at least severalweeks).

Another essential objective of the invention is to provide a medium forthe detection/identification of microorganisms, especially bacteria oryeasts, with an enzymatic activity selected from esterase and/or osidaseand/or peptidase and/or sulfatase and/or phosphatase activities which isnot in the form of a dry powder that has to be reconstituted with aliquid in order to reconstitute a liquid or gelled medium, but whichexists directly in ready-to-use forms.

Another essential objective of the invention is to provide a medium forthe detection/identification of microorganisms, especially bacteria oryeasts, with an enzymatic activity selected from esterase and/or osidaseand/or peptidase and/or sulfatase and/or phosphatase activities which iseconomic, especially due to the fact that it comprises reduced amountsof one or more chromogenic or fluorogenic enzyme substrates, which arecharacteristically expensive.

Another essential objective of the invention is to provide a method ofobtaining the above-mentioned detection/identification medium which issimple to carry out and economic.

Another essential objective of the present invention is to provide amethod for the detection/identification of strains with an enzymaticactivity selected from esterase, osidase and/or peptidase and/orsulfatase and/or phosphatase activities which is easy to carry out(routine tests), economic (amount of reagent, handling, label, speed,etc.), reliable, sensitive, specific and reproducible.

Another essential objective of the present invention is to propose theuse of a novel stabilizing additive in media for thedetection/identification of microorganisms, especially bacteria oryeasts, with an enzymatic activity selected from esterase and/or osidaseand/or peptidase and/or sulfatase and/or phosphatase activities, saidactivity being revealed as a result of a hydrolysis reaction whichreleases a dye or a fluorescent product.

These and other objectives are achieved by the present invention, whichrelates first and foremost to a medium for the detection/identificationof microorganisms, especially bacteria and/or yeasts, with an enzymaticactivity selected from esterase and/or osidase and/or peptidase and/orsulfatase and/or phosphatase activities that is of the type comprisingespecially a reaction medium, particularly a culture medium, and atleast one chromogenic or fluorogenic esterase and/or osidase and/orpeptidase and/or sulfatase and/or phosphatase substrate, with theexclusion of substrates comprising a4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]quinolinium-1-(propan-3-ylcarboxylicacid) cation and an anion, this detection/identification being basedessentially on revealing the presence of the esterase and/or osidaseand/or peptidase and/or sulfatase and/or phosphatase activities, . . .said medium:

-   -   is in a stable, ready-to-use liquid or gel form; and    -   contains:        -   at least one fatty acid sorbitan ester (FASE) or at least            one fatty acid (FA) or an FASE/FA mixture (comprising at            least one fatty acid sorbitan ester and at least one fatty            acid) as an emulsifying stabilizer, and        -   optionally at least one solvent (S).

A medium for the detection/identification of microorganisms, especiallybacteria and/or yeasts, with an enzymatic activity selected fromesterase and/or osidase and/or peptidase and/or sulfatase and/orphosphatase activities, according to the invention, can be obtained fromthe media presented in patent documents WO-95/04157 and WO-00/53799described above.

The medium according to the invention has the advantage that it is readyto use and is in a liquid or semiliquid (gel) form without presenting aproblem in terms of stability. In fact, the esterase and/or osidaseand/or peptidase and/or sulfatase and/or phosphatase substrates presentin this medium, even though they are known for their tendency to degraderelatively rapidly, are stabilized through the presence of the FASE, FAor FASE/FA emulsifying stabilizer additive.

This additive, optionally associated with the solvent S, brings afurther advantage, namely allowing an excellent dissolution of theesterase and/or osidase and/or peptidase and/or sulfatase and/orphosphatase substrates, which are traditionally reluctant to dissolve.This makes it possible to obtain detection/identification media that aretranslucent before inoculation, thereby making it easier to read thecoloration or fluorescence results.

This FASE, FA or FASE/FA additive is relatively inexpensive and makes itpossible to simplify the procedure and to reduce the amount ofsubstrates used (stabilization), resulting in a definite saving ineconomic terms.

Finally, the means according to the invention give rise to animprovement in the biological activity of the afore-mentionedsubstrates, resulting in a more intense coloration of the targetcolonies and a better specificity.

It is to the inventors' credit to have selected this particular class ofemulsifying stabilizers, particularly FASE, which surprisingly andunexpectedly afford properties of stability, translucence, biologicalactivity, sensitivity, reliability and specificity that are altogetherapposite in the microbiological analysis by biochemical means accordingto the invention.

According to one noteworthy characteristic of the invention, thedetection/identification medium to which it relates is obtained bymixing at least one stock solution of the substrate in the solvent Swith FASE, FA or an FASE/FA mixture, and with at least some of theconstituents of the culture medium.

In fact, it appears to be important according to the invention tosolubilize the esterase and/or osidase and/or peptidase and/or sulfataseand/or phosphatase substrate in the solvent S in the presence of one ormore FASE or FA or an FASE/FA mixture as defined above. This mixture ofFASE, FA or FASE/FA substrate and solvent S is then incorporated into atleast part of the culture medium, which is preferably in a supercooledgelled form. It has been found that this operating characteristic makesit possible to optimize the advantageous results produced by the meansaccording to the invention.

Preferably, the FASE is selected from the group comprising:

-   -   polyethoxylated sorbitan monolaurate containing 20 units of        ethylene oxide (EO)—TWEEN® 20-;    -   polyethoxylated sorbitan monopalmitate (20 EO)—TWEEN® 40-;    -   polyethoxylated sorbitan monostearate (20 EO)—TWEEN® 60-;    -   polyethoxylated sorbitan tristearate (20 EO)—TWEEN® 65-;    -   polyethoxylated sorbitan monooleate (20 EO)—TWEEN® 80-;    -   polyethoxylated sorbitan sesquioleate (20 EO)—TWEEN® 83-;    -   polyethoxylated sorbitan trioleate (20 EO)—TWEEN® 85-; and    -   mixtures thereof,        and the FA is selected from the group comprising C4–C20        saturated or unsaturated fatty acids, preferably C6–C11        saturated or unsaturated fatty acids and particularly preferably        C8–C9 fatty acids, and mixtures thereof.

The products particularly selected according to the invention aresorbitan esters that are widely used in the food and cosmetic industriesas non-ionic emulsifiers, but hitherto they have never been employed inmicrobiological detection/identification media as stabilizers foresterase and/or osidase and/or peptidase and/or sulfatase and/orphosphatase substrates.

The Hydrophilic-Lipophilic Balances (HLB) of the above-mentioned FASEare respectively 8.6, 6.7, 4.7, 2.1, 4.3, 3.7 and 1.8.

In one advantageous variant of the invention, the emulsifyingstabilizer(s) described above, in particular the FASE(s), can beassociated with at least one synergistic co-agent, preferably at leastone anionic surfactant and particularly preferably7-ethyl-2-methyl-4-undecyl hydrogensulfate or its salts, moreparticularly its sodium salts (TERGITOL-4®).

The use of such a synergistic co-agent makes it easier to reveal thepresence of the enzymatic activity in the target bacteria. Said co-agentis an excellent complement to the emulsifying stabilizer(s) andparticularly the FASE. It makes it possible to improve the selectivityand the detection of the esterase and/or osidase and/or peptidase and/orsulfatase and/or phosphatase activities in the target microorganismswithout detracting from the expression of other enzymatic activitieswhich might possibly be used to reveal the presence of the targetmicroorganisms in question. Without wishing to be bound by theory, it isassumed that the synergistic co-agent TERGITOL-4® has an action whichfavors the penetration of the chromogenic or fluorogenic esterase and/orosidase and/or peptidase and/or sulfatase and/or phosphatase substratesor the excretion of the enzymes across the membranes of the cells of thetarget microorganisms, thereby optimizing the accessibility of thesesubstrates in respect of the enzymes whose activity is sought. Thismakes it possible to reduce the amount of substrates used and derive adefinite economic advantage therefrom.

Even more preferably, the combination of TWEEN® and TERGITOL-4® seems tobe totally effective in the detection/identification medium according tothe invention.

The chromogenic or fluorogenic substrate, consisting of a target partfor the enzyme and a chromophoric or fluorophoric part, isadvantageously chosen from substrates whose target part is selected fromthe group comprising the following in particular:

-   -   glycosides consisting of mono-, di- and/or polysaccharide units        linked in the α- or β-position to the hydroxyl group of the        chromophoric or fluorophoric part;    -   α-amino acids or peptides;    -   organic acids such as —O—CO(CH₂)_(n)—CH₃, where n is between 0        and 20; and    -   a sulfate, a phosphate, a pyrosulfate, a pyrophosphate and a        phosphodiester,        and whose chromophoric or fluorophoric part is selected from the        group comprising the following in particular:    -   quinones/anthraquinones and derivatives, especially        dihydroxyanthraquinone (alizarin);    -   amino- and hydroxycoumarins and derivatives;    -   fluoresceins and derivatives; and    -   indoxyls and derivatives.

Examples of esterase substrates which may be mentioned are chromogenicester substrates derived from indole, especially 5-bromo-3-indolylnonanoate, 6-chloro-3-indolyl nonanoate or 5-bromo-3-indolyl decanoate.

2-Alizarin octanoate may be mentioned as an example of a chromogenicesterase substrate derived from anthraquinone.

Examples of osidase substrates which may be mentioned are chromogenicosidase substrates derived from indole, especially6-chloro-3-indolyl-β-galactoside,5-bromo-4-chloro-3-indolyl-β-N-acetylglucosaminide or5-bromo-6-chloro-α-glucoside.

2-Alizarin-β-glucuronide may be mentioned as an example of a chromogenicosidase substrate derived from anthraquinone.

Fluorogenic osidase substrates which may be mentioned are substratesderived from hydroxycoumarin, especially4-methylumbelliferone-β-glucoside.

Substrates derived from indole may be mentioned as chromogenic peptidasesubstrates.

Fluorogenic peptidase substrates are especially substrates derived fromhydroxycoumarin, such as alanylaminomethylcoumarin.

Sulfatase substrates which may be mentioned are chromogenic substratesderived from indole, particularly 5-bromo-4-chloro-3-indolyl sulfate.4-Methylumbelliferone sulfate is an example of a fluorogenic sulfatasesubstrate.

In an equivalent manner, phosphatase substrates which may be mentionedare chromogenic substrates derived from indole, such as5-bromo-4-chloro-3-indolyl phosphate. 4-Methylumbelliferone phosphate isan example of a fluorogenic phosphatase substrate.

The solvent S is an auxiliary for solubilizing the enzyme substrate ofinterest, particularly a chromogenic enzyme substrate. It alsocomplements the action of the FASE, FA or FASE/FA emulsifyingstabilizer. It is therefore preferably a constituent of the mediumaccording to the invention.

According to an advantageous provision of the invention, the solvent Sis selected from the group comprising:

-   -   alcohols, preferably methanol, ethanol and methoxyethanol;    -   amides, preferably dimethylformamide (DMF);    -   sulfur-containing solvents, preferably dimethyl sulfoxide        (DMSO);    -   aqueous solvents, preferably water and buffered water; and    -   mixtures thereof.

In practice, the solvents preferably used are methanol, DMF and DMSO.

It has been found altogether advantageous according to the invention forthe proportion by weight of fatty acid sorbitan ester(s) (FASE) tosolvent (S) to be between 20:80 and 80:20, preferably between 30:70 and70:30 and particularly preferably 40:60 or 60:40.

Still from the quantitative point of view, it is preferable for theconcentration of (FASE) in the medium to be defined as follows (in % byweight):

0.1 ≦ [FASE] ≦ 10, preferably 0.5 ≦ [FASE] ≦ 5, and particularlypreferably 1.5 ≦ [FASE] ≦ 3.5.

The amounts of chromogenic or fluorogenic substrate used are such thatits concentration in the medium is defined as follows (in mg/l):

 1 ≦ [substrate] ≦ 2000, preferably  5 ≦ [substrate] ≦ 1500, andparticularly preferably 25 ≦ [substrate] ≦ 1000.

The amounts of substrate used in the solution of FASE and solvents S aresuch that its concentration in the medium is defined as follows (ing/l):

0.5 ≦ [substrate in stock solution] ≦ 2000, preferably   1 ≦ [substratein stock solution] ≦ 1000, and particularly   2 ≦ [substrate in stocksolution] ≦ 200. preferably

As regards the culture medium present in the detection/identificationmedium, it can be specified that it is selected from the groupcomprising:

-   -   selective media of the type comprising MacConkey, Columbia ANC,        PALCAM and Sabouraud gentamycin-chloramphenicol media,        preferably MacConkey medium; and    -   non-selective media of the type comprising Columbia +/− blood,        trypcase soya, nutrient gelose and Sabouraud media, preferably        Columbia medium.

In practice, those skilled in the art will choose the culture mediumaccording to the target bacteria and according to perfectly knowncriteria with which they are familiar.

Without implying a limitation, it is found that the medium according tothe invention is particularly suitable for the detection/identificationof microorganisms of medical or industrial interest, especially those ofthe genus Salmonella, Pseudomonas, Listeria, Staphylococcus,Enterococcus or Candida.

In the case of the detection of bacteria of the genus Salmonella,MacConkey medium, for example, will be chosen as the culture medium.

Furthermore, the medium according to the invention can optionallycontain other additives such as one or more other enzyme substrates, forexample chromogenic or fluorogenic enzyme substrates, peptones, one ormore growth factors, carbohydrates, one or more selective agents,buffers and one or more gelling agents.

The medium according to the invention is in the form of a liquid or gelthat is ready to use, i.e. ready for inoculation in a tube or flask oron Petri dishes.

The medium according to the invention can be stored in its containersfor several weeks at 40° C. in liquid or gel form.

According to another of its features, the present invention furtherrelates to a method of obtaining the medium as defined above,characterized in that it consists essentially in:

-   -   preparing at least one stock solution of the chromogenic or        fluorogenic esterase and/or osidase and/or peptidase and/or        sulfatase and/or phosphatase substrate in the solvent S and of        at least one FASE, FA or FASE/FA mixture,    -   adding this solution and any other additives to the culture        medium, and    -   homogenizing the whole.

The stock solution is prepared separately by successively incorporatingthe substrate, the solvent S and the FASE, FA or FASE/FA mixture,optionally containing co-additives. The products and the amounts usedare as defined above.

After homogenization, the stock solution is added to the supercooledgelled culture medium which has been regenerated in water beforehand.The medium can also be a non-gelled liquid medium, for example anutrient broth.

Mixing of the culture medium and the stock solution gives the liquid orgelled detection medium ready for inoculation.

According to yet another of its features, the invention further relatesto a method for the detection/identification of strains with esteraseand/or osidase and/or peptidase and/or sulfatase and/or phosphataseactivity which consists in:

-   -   inoculating the detection/identification medium as defined        above, either as a product per se or as a product obtained by        the method also described above, with the sample containing the        target bacteria to be analyzed;    -   incubating the inoculated medium under appropriate conditions        known to those skilled in the art; and    -   reading/interpreting the colorations or fluorescences of the        colonies which have developed after incubation, for example in        an oven at 37° C., these colorations revealing hydrolysis of the        chromogenic or fluorogenic esterase and/or osidase and/or        peptidase and/or sulfatase and/or phosphatase substrate by the        target bacteria.

Finally, the invention further relates to the use of fatty acid sorbitanester(s) (FASE), fatty acids (FA) or an FASE/FA mixture as anemulsifying stabilizer for a medium (liquid or gel) for thedetection/identification of microorganisms, especially bacteria oryeasts, with an enzymatic activity selected from esterase and/or osidaseand/or peptidase and/or sulfatase and/or phosphatase activities, thismedium comprising especially a reaction medium and at least onechromogenic or fluorogenic esterase and/or osidase and/or peptidaseand/or sulfatase and/or phosphatase substrate, with the exclusion ofsubstrates comprising a4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)vinyl]quinolinium-1-(propan-3-yl)carboxylateand an anion, optionally in association with at least one solvent S forthe chromogenic or fluorogenic esterase and/or osidase and/or peptidaseand/or sulfatase and/or phosphatase substrate present in the medium.

The Examples which follow will provide a clearer understanding of theinvention and make it possible to assess all its advantages as well asits diverse embodiments and modes of implementation.

EXAMPLES Example 1 Testing the Solubility of the Indoxyl-Based EsteraseSubstrate 5-bromo-3-indolyl Nonanoate (Chromogenic Esterase Substrate)in a Gelled Medium

Different stock solutions of substrate are prepared in methanol in thepresence of no. 3 bile salts (bioMérieux). Different volumes of thesesolutions are then added to a supercooled gelled medium, namelyMacConkey medium (regenerated in water at a concentration of 50 g/l).

The protocol and the appearance of the media are collated in Table 1below.

TABLE 1 Medium 1 2 3 4 5 Substrate concentration 100 g/l 50 g/l 25 g/l12.5 g/l 6.25 g/l of the stock solution Final substrate 500 mg/lconcentration of the medium Bile salt concentration 5 g/l 2.5 g/l of thestock solution Total bile salt 7.5 g/l 4 g/l concentration of the mediumMethanol concentration 0.5% 1% 2% 4% 8% of the medium Observation of theprecipitate precipitate slight milky milky medium poured into in theform in the form precipitate: medium medium dishes of coarse of smallermilky grains grains than medium in medium 1

The most optically homogeneous media are 3, 4 and 5, being the media inwhich the methanol concentration is greatest. However, these media havea “milky” appearance, so it would seem that the esterase substrate is inthe form of an emulsion.

Example 2 Improving the Solubilization of the Esterase Substrate byAdding Tween® 20 (FASE)

Two different stock solutions of substrate are prepared in methanol asthe solvent S, one in the presence of no. 3 bile salts and the other inthe presence of no. 3 bile salts and Tween® 20 [polyethoxylated sorbitanmonolaurate containing 20 units of ethylene oxide (EO)]. A volumecorresponding to a final concentration of 500 mg/l of substrate is thenadded to a supercooled gelled culture medium, namely MacConkey medium.The protocol and the appearance of the media are collated in Table 2below.

TABLE 2 Medium 1 2 Substrate concentration of 100 g/l 40 g/l the stocksolution Final substrate concentration 500 mg/l 500 mg/l of the mediumBile salt concentration of 5 g/l 5 g/l the stock solution Total bilesalt concentration 6.5 g/l 6.5 g/l of the medium Concentration ofTween ® 0%  60% 20 added to the stock solution of substrateConcentration of Tween ® 0% 2.5% 20 added to the medium Observation ofthe medium milky translucent poured into dishes medium medium

Medium 2 has a better optical homogeneity than medium 1. The esterasesubstrate is either completely dissolved or in the form of an emulsionwhose micellar particles are no longer visible to the eye.

Example 3 Biological Testing of the Two Media Described in the PreviousExample

Microorganisms originating from the Applicant's collection wereinoculated onto each of the two media described above from a 0.5McFarland suspension by isolation in three quadrants. The dishes wereincubated at 37°C. for 48 hours. The colonies formed were examinedvisually after 24 and 48 hours of incubation. The coloration of thesecolonies and the intensity of this coloration were noted. The resultsare shown in Table 3 below.

TABLE 3 incubation medium 1 medium 2 Strain time color intensity colorintensity Salmonella 24 h gray 1.5 gray 3 typhimurium 10 48 h gray 1.5gray 3.5 Salmonella 24 h gray 1 gray 3 paratyphi A 152 48 h gray 1.5gray 3.5 Proteus 24 h — — — — vulgaris 15 48 h — — — — Serratia 24 hgray 1.5 gray 3 marcescens 13 48 h gray 1 gray 4 intensity ofcoloration: arbitrary scale; —: absence of coloration or intensity

The medium containing Tween® 20 therefore makes it possible to obtainmore intense colorations for the strains of microorganisms exhibiting anesterase activity. The substrate is therefore more widely used eitherbecause it dissolves better or because it is more readily available. Inview of the difference in intensity between the two media, it seems tobe possible, in the presence of Tween® 20, to reduce the concentrationof substrate used and hence to lower the cost incurred by the use ofthis type of substrate.

Example 4 Testing this Mode of Dissolution for Another IndoxylDerivative (Chromogenic Esterase Substrate) on Gram-positive andGram-negative Bacterial Species and Yeasts

A 40 g/l stock solution of 5-bromo-4-chloro-3-indolyl octanoate(chromogenic esterase substrate) was prepared in a mixture of methanol(40%) and Tween® 20 (60%). A volume corresponding to a finalconcentration of 500 mg/l of substrate was then added to a supercooledgelled medium, namely a medium of the Columbia type. This medium waspoured into Petri dishes.

Microorganisms originating from the Applicant's collection wereinoculated from a 0.5 McFarland suspension by isolation in threequadrants. The dishes were then incubated at 37° C. for 48 hours. Thecolonies formed were examined visually after 24 and 48 hours ofincubation. The coloration of these colonies and the intensity of thiscoloration were noted. The results are shown in Table 4 below.

TABLE 4 incubation medium 1 Strain time color intensity Listeria 24 hturquoise 2.5 monocytogenes 023 48 h turquoise 2.5 Listeria 24 h — —innocua 029 48 h turquoise 0.5 Staphylococcus 24 h turquoise 2.5 aureus276 48 h turquoise 3 Candida 24 h turquoise 0.5 albicans 033 48 hturquoise 3 Staphylococcus 24 h turquoise 2 epidermidis 009 48 hturquoise 2 Salmonella spp. 24 h turquoise 2 017 48 h turquoise 3Proteus 24 h — — vulgaris 015 48 h — — intensity of coloration:arbitrary scale; —: absence of coloration or intensity

In the presence of Tween 20, it is therefore possible to reveal theexpression of an esterase activity in all microorganisms that exhibitit, independently of the group to which they belong. Furthermore, thismode of use is independent of the indoxyl derivative (chromogenicesterase substrate) studied.

Example 5 Replacing Methanol with Other Solvents in the Presence ofTween 20 and in the Presence of Bile Salts

The methanol used in the previous Examples was replaced with two othersolvents, namely dimethyl sulfoxide (DMSO) and dimethylformamide (DMF).The three solvents were tested in the presence of Tween 20 and in thepresence or absence of bile salts at a final concentration of 6.5 g/l inthe medium. Table 5 below shows the composition of the six media tested.

TABLE 5 Medium 1 2 3 4 5 6 Solvent S of the methanol DMSO DMF methanolDMSO DMF stock solution (SS) % of solvent S in 40% the SS % of Tween ®20 60% added to the SS of substrate Bile salt 6.5 g/l 0 g/lconcentration of the medium Final substrate 500 mg/l concentration ofthe medium

The activity of the substrate was verified in the same way as in theprevious Example, i.e. in the presence of microorganisms inoculated ontoa MacConkey gelose medium containing the esterase substrate dissolved inone of the solvents mentioned above. The results are shown in Table 6below.

TABLE 6 Medium 1 2 3 4 5 6 Strain IT C I C I C I C I C I C I Salmonella24 gray 2 gray 2 gray 2 gray 2 gray 2 gray 2 typhimurium 10 48 gray 2.5gray 2.5 gray 2.5 gray 2.5 gray 2.5 gray 2.5 Salmonella 24 gray 2 gray2.5 gray 2 gray 2 gray 2.5 gray 2 arizonae 15 48 gray 2.5 gray 3 gray2.5 gray 2.5 gray 3 gray 2.5 Pseudomonas 24 gray 2 gray 2 gray 1 gray 2gray 2 gray 1 aeruginosa 15 48 gray 2.5 gray 4 gray 3.5 gray 3 gray 4gray 4 Proteus 24 — — — — — — — — — — — — vulgaris 15 48 — — — — — — — —— — — — Serratia 24 gray 2.5 gray 2 gray 2 gray 2.5 gray 2 gray 2marcescens 38 48 gray 2.5 gray 2 gray 2 gray 2.5 gray 2 gray 2 I:intensity of coloration (arbitrary scale); C: color; IT: incubation timein hours; —: colorless

It is therefore possible to use different solvents to dissolve theesterase substrate when Tween 20 is present. Furthermore, the additionof bile salts makes no difference, whether in terms of solubility or interms of biological results, irrespective of the solvent tested. It isconcluded that the simplest mode of dissolution is to mix a solvent andTween® 20 without the additional incorporation of bile salts.

Example 6 Testing Different Solvent S/Tween 20 Ratios

Different stock solutions of substrate are prepared in DMSO in thepresence of Tween® 20. Each stock solution corresponds to a differentDMSO/Tween® 20 ratio. A volume corresponding to a final concentration of500 mg/l of substrate is then added to a supercooled gelled medium,namely MacConkey medium. Table 7 below shows the composition of the sixmedia tested.

TABLE 7 Medium 1 2 3 4 5 6 % of DMSO in the 10% 20% 30% 40% 50% 60%stock solution % of Tween ® 20 added 90% 80% 70% 60% 50% 40% to thestock solution of substrate

Microorganisms originating from the Applicant's collection wereinoculated onto each of the six media described above from a 0.5McFarland suspension by isolation in three quadrants. The dishes wereincubated at 37° C. for 48 hours. The colonies formed were examinedvisually after 24 and 48 hours of incubation. The coloration of thesecolonies and the intensity of this coloration were noted. The resultsare shown in Table 8 below.

TABLE 8 Medium 1 2 3 4 5 6 Strain IT C I C I C I C I C I C I Salmonella24 gray 2.5 gray 2.5 gray 2 gray 3 gray 3 gray 3 typhimurium 10 48 gray3 gray 3 gray 2.5 gray 3.5 gray 3 gray 3.5 Salmonella 24 gray 2.5 gray2.5 gray 3 gray 3.5 gray 3 gray 3.5 arizonae 15 48 gray 2.5 gray 2.5gray 3 gray 3.5 gray 3 gray 3.5 Serratia 24 gray 3 gray 3 gray 3 gray 4gray 3.5 gray 3.5 marcescens 045 48 gray 4 gray 4 gray 4 gray 4 gray 4gray 4 Hafnia alvei 24 — — — — — — — — — — — — 025 48 — — — — — — — — —— — — Pseudomonas 24 gray 2 gray 2 gray 3 gray 3 gray 3 gray 3aeruginosa 165 48 gray 2 gray 2 gray 2 gray 3 gray 3 gray 3 Salmonella24 gray 1.5 gray 1.5 gray 2 gray 3 gray 2.5 gray 3 paratyphi A 006 48gray 2 gray 2 gray 2 gray 3 gray 3 gray 3 Salmonella 24 gray 2 gray 2gray 2 gray 3 gray 2 gray 3 typhi 118 48 gray 2 gray 2 gray 2 gray 3gray 2 gray 3 I: intensity of coloration (arbitrary scale); C: color;IT: incubation time in hours; —: colorless

The six ratios tested make it possible to obtain a good substratesolubility and to reveal hydrolysis of the substrate with veryacceptable intensities of coloration. Maximum intensities of colorationare obtained between the ratios 40% DMSO/60% Tween® 20 and 60% DMSO/40%Tween® 20.

Example 7 Testing the Method of Solubilization on an Esterase SubstrateHaving a Marker Other than an Indoxyl

The substrate tested is 1,2-dihydroxyanthraquinone octanoate (2-alizarinoctanoate). Different stock solutions of substrate are prepared withDMF, DMSO, methanol or methoxyethanol as solvent and in the presence ofTween® 20 in all cases. Table 9 below shows the appearance of the stocksolutions according to their composition.

TABLE 9 Solvent of the stock solution methanol DMSO DMF methoxyethanol %of solvent in 40% the stock solution % of Tween ® 20 60% added to thestock solution of substrate Appearance of the precipitate precipitatetotal total stock solution dissolution dissolution

A volume corresponding to a final concentration of 100 mg/l of substrateis then added to a supercooled gelled medium, namely MacConkey medium.Iron citrate at a final concentration of 50 mg/l was also added to themedium so that the esterase activity could be revealed in the presenceof an alizarin-based substrate. Only one of the stock solutionsexhibiting total dissolution of the substrate was tested (stock solutionin DMF). Microorganisms originating from the Applicant's collection wereinoculated onto each of the two media described above from a 0.5McFarland suspension by isolation in three quadrants. The dishes wereincubated at 37° C. for 48 hours. The colonies formed were examinedvisually after 24 and 48 hours of incubation. The coloration of thesecolonies and the intensity of this coloration were noted. The resultsare shown in Table 10.

TABLE 10 incubation medium with DMF Strain time color intensitySalmonella typhimurium 24 h mauve 1 10 48 h mauve 2 Salmonellaenteritidis 24 h mauve 1 036 48 h mauve 3 Salmonella arizonae 24 h mauve1 018 48 h mauve 2 Escherichia coli 24 h — — 002 48 h — — Serratiamarcescens 24 h mauve 3.5 045 48 h mauve 3.5 Staphylococcus aureus 24 hmauve 3 033 48 h mauve 4 Pseudomonas aeruginosa 24 h mauve 2 165 48 hmauve 2 Salmonella gallinarum 24 h mauve 0.5 500 48 h mauve 3 intensityof coloration: arbitrary scale; —: absence of coloration or intensity

This mode of dissolution is therefore not specific for indoxyl esters,but can be used especially with other families of enzyme substrates.

Example 8 Testing Different FASE Derived from Tween® 20 for theSolubilization of the Esterase Substrate 5-bromo-3-indolyl Nonanoate

Different stock solutions of substrate are prepared in DMSO in thepresence of Tween® 20, Tween® 80 (polyethoxylated sorbitan monooleate),Tween® 60 (polyethoxylated sorbitan monostearate) or Tween® 65(polyethoxylated sorbitan tristearate). A volume corresponding to afinal concentration of 500 mg/l of substrate is then added to asupercooled gelled medium, namely MacConkey medium. Table 11 shows thecomposition of the media tested.

TABLE 11 Medium 1 2 3 4 % of DMSO in the stock solution 40% 40% 40% 40%% of Tween 20 added to the 60% — — — stock solution of substrate % ofTween 60 added to the — 60% — — stock solution of substrate % of Tween65 added to the — — 60% — stock solution of substrate % of Tween 80added to the — — — 60% stock solution of substrate

Microorganisms originating from the Applicant's collection wereinoculated onto each of the six media described above from a 0.5McFarland suspension by isolation in three quadrants. The dishes wereincubated at 37° C. for 48 hours. The colonies formed were examinedvisually after 24 and 48 hours of incubation. The coloration of thesecolonies and the intensity of this coloration were noted. The resultsare shown in Table 12.

TABLE 12 Medium 1 2 3 4 Strain IT C I C I C I C I Salmonella 24 h gray2.5 gray 2 — — gray 2 typhimurium 10 48 h gray 3 gray 2 — — gray 2Salmonella 24 h gray 2.5 gray 1 — — gray 1 arizonae 15 48 h gray 3 gray2 — — gray 3 Salmonella 24 h gray 1.5 gray 1 — — gray 1 paratyphi A 00648 h gray 3.5 gray 2 — — gray 3 Salmonella 24 h gray 2 gray 1 — — gray 1typhi 118 48 h gray 2.5 gray 2 — — gray 2 Pseudomonas 24 h gray 3 gray0.5 — — gray 4 aeruginosa 165 48 h gray 3 gray 2 — — gray 4 Hafnia alvei24 h — — — — — — — — 025 48 h — — — — — — — — I: intensity of coloration(arbitrary scale); C: color; IT: incubation time; —: colorless

Note: Different Tween®/solvent ratios were prepared for each Tween®tested. However, only one ratio is listed in this Example because theresults were very similar, irrespective of the ratio studied.

Tween® 20, Tween® 80 and Tween® 60 make it possible to obtain a goodsubstrate solubility and to reveal hydrolysis of the substrate with veryacceptable intensities of coloration.

Example 9 Comparative Stability of Gelled Media Containing the EsteraseSubstrate 5-bromo-3-indolyl Nonanoate Solubilized or Not Solubilized inthe Presence of Tween® 20

Two stock solutions of substrate were prepared, one having aconcentration of 40 g/l in methanol in the presence of 6.5 g/l of no. 3bile salts, and the other having a concentration of 40 g/l in a mixtureof DMSO (40%) and Tween® 20 (60%). A volume corresponding to a finalconcentration of 500 mg/l of substrate was then added to a supercooledgelled medium, namely MacConkey medium. These two media were poured intoPetri dishes of diameter 90 mm and stored at +4° C. for 8 weeks. Afterone week, two weeks, four weeks and eight weeks, two media like thosedescribed above were prepared for immediate use.

Microorganisms originating from the Applicant's collection wereinoculated from a 0.5 McFarland suspension, by isolation in threequadrants, onto the two media prepared for immediate use and the twomedia stored at +4° C. The dishes were incubated at 37° C. for 48 hours.The colonies formed were examined visually after 24 and 48 hours ofincubation. The coloration of these colonies and the intensity of thiscoloration were noted. The results are shown in the Tables below.

TABLE 13 stored for stored for fresh 1 week fresh 1 week methanolmethanol DMSO DMSO Medium bile salts bile salts Tween 20 Tween 20 StrainIT C I C I C I C I Salmonella typhimurium 24 h gray 0.5 gray 0.5 gray 3gray 3 10 48 h gray 1.5 gray 1.5 gray 4 gray 4 Salmonella arizonae 24 hgray 1 gray 1 gray 3 gray 3 15 48 h gray 1.5 gray 1.5 gray 4 gray 4Salmonella paratyphi A 24 h — — — — gray 2 gray 2 006 48 h gray 0.5 gray0.5 gray 4 gray 4 Salmonella typhi 24 h gray 0.5 gray 0.5 gray 3 gray 3118 48 h gray 1.5 gray 1.5 gray 4 gray 4 Pseudomonas aeruginosa 24 hgray 1 gray 1 gray 2 gray 2 165 48 h gray 1 gray 1 gray 3 gray 3 Hafniaalvei 24 h — — — — — — — — 025 48 h — — — — — — — —

TABLE 14 stored for stored for fresh 2 weeks fresh 2 weeks methanolmethanol DMSO DMSO Medium bile salts bile salts Tween 20 Tween 20 StrainIT C I C I C I C I Salmonella 24 h gray 0.5 gray 0.5 gray 3 gray 3typhimurium 10 48 h gray 1.5 gray 1.5 gray 4 gray 4 Salmonella 24 h gray1 gray 0.1 gray 3 gray 3 arizonae 15 48 h gray 1.5 gray 0.5 gray 4 gray4 Salmonella 24 h — — — — gray 2 gray 2 paratyphi A 006 48 h gray 0.5gray 0.1 gray 4 gray 4 Salmonella 24 h gray 0.5 — — gray 3 gray 3 typhi118 48 h gray 1.5 gray 0.5 gray 4 gray 4 Pseudomonas 24 h gray 1 gray 1gray 2 gray 2 aeruginosa 165 48 h gray 1 gray 1 gray 3 gray 3 Hafniaalvei 24 h — — — — — — — — 025 48 h — — — — — — — —

TABLE 15 stored for stored for fresh 4 weeks fresh 4 weeks methanolmethanol DMSO DMSO Medium bile salts bile salts Tween 20 Tween 20 StrainIT C I C I C I C I Salmonella 24 h gray 0.5 — — gray 3 gray 3typhimurium 10 48 h gray 1.5 gray 0.5 gray 4 gray 4 Salmonella 24 h gray1 gray 0.1 gray 3 gray 3 arizonae 15 48 h gray 1.5 gray 0.5 gray 4 gray4 Salmonella 24 h — — — — gray 2 gray 2 paratyphi A 006 48 h gray 0.5gray 0.1 gray 4 gray 4 Salmonella 24 h gray 0.5 — — gray 3 gray 3 typhi118 48 h gray 1.5 gray 0.5 gray 4 gray 4 Pseudomonas 24 h gray 1 gray0.5 gray 2 gray 2 aeruginosa 165 48 h gray 1 gray 1 gray 3 gray 3 Hafniaalvei 24 h — — — — — — — — 025 48 h — — — — — — — —

TABLE 16 stored for stored for fresh 8 weeks fresh 8 weeks methanolmethanol DMSO DMSO Medium bile salts bile salts Tween 20 Tween 20 StrainIT C I C I C I C I Salmonella 24 h gray 0.5 — — gray 3 gray 2.5typhimurium 10 48 h gray 1.5 gray 0.5 gray 4 gray 3.5 Salmonella 24 hgray 1 — — gray 3 gray 3 arizonae 15 48 h gray 1.5 gray 0.5 gray 4 gray3 Salmonella 24 h — — — — gray 2 gray 1.5 paratyphi A 006 48 h gray 0.5— — gray 4 gray 3 Salmonella 24 h gray 0.5 — — gray 3 gray 2 typhi 11848 h gray 1.5 gray 0.5 gray 4 gray 3 Pseudomonas 24 h gray 1 gray 0.5gray 2 gray 2 aeruginosa 165 48 h gray 1 gray 1 gray 3 gray 3 Hafniaalvei 24 h — — — — — — — — 025 48 h — — — — — — — — I: intensity ofcoloration (arbitrary scale); C: color; IT: incubation time; —:colorless

The medium containing the substrate dissolved in a mixture of methanoland bile salts exhibits a very substantial drop in the expression ofesterase activity after storage for 2 weeks. This drop is amplified overtime. The medium containing the substrate dissolved in a mixture of DMSOand Tween 20 exhibits a constant expression of esterase activity overtime for up to at least six weeks. After eight weeks there is a slightbut not prohibitive drop in the expression of esterase activity. Tween20 therefore makes it possible to stabilize the esterase substrate andmakes it easier to store and use culture media containing this type ofsubstrate.

Note: In the above Examples, the numbers following the strainscorrespond to the number of each strain referenced in the Applicant'scollection. The intensity of coloration corresponds to an arbitraryscale defined as follows:

0 no activity 0.1 trace of coloration 0.5 very pale coloration 1distinct coloration of low intensity 2 clear coloration of mediumintensity 3 intense coloration 4 very intense coloration

Example 10 Comparative Stability of Gelled Media Containing the OsidaseSubstrate 6-chloro-3-indolyl-β-N-acetylglucosaminide Solubilized or NotSolubilized in the Presence of Tween® 20

Two stock solutions of substrate were prepared, one having aconcentration of 50 g/l in DMF and the other having a concentration of50 g/l in a mixture of DMF (40%) and Tween® 20 (60%). A volumecorresponding to a final concentration of 175 mg/l of substrate was thenadded to a supercooled gelled medium, namely Columbia medium notsupplemented with blood. These two media were poured into Petri dishesof diameter 90 mm and stored at +4° C. for 8 weeks. After three weeks,six weeks, nine weeks and twelve weeks, two media like those describedabove were prepared for immediate use.

Microorganisms originating from the Applicant's collection wereinoculated from a 0.5 McFarland suspension, by isolation in threequadrants, onto the two media prepared for immediate use and the twomedia stored at +4° C. The dishes were incubated at 37° C. for 48 hours.The colonies formed were examined visually after 24 and 48 hours ofincubation. The coloration of these colonies and the intensity of thiscoloration were noted. The results are shown in the Tables below.

TABLE 17 stored for stored for fresh 3 weeks fresh 3 weeks DMF DMFMedium DMF DMF Tween 20 Tween 20 Strain IT C I C I C I C I Listeria 24 hM 2 M 1.5 M 2 M 2 innocua 36 48 h M 3 M 3 M 3 M 3 Listeria 24 h M 1.5 M1 M 1.5 M 1.5 ivanovii 18 48 h M 2.5 M 2.5 M 2.5 M 2.5 Listeria 24 h M 2M 1.5 M 2 M 2 monocytogenes 23 48 h M 3 M 3 M 3 M 3 Staphylococcus 24 h— — — — — — — — aureus 20 48 h — — — — — — — — Pseudomonas 24 h — — — —— — — — aeruginosa 165 48 h — — — — — — — —

TABLE 18 stored for stored for fresh 6 weeks fresh 6 weeks DMF DMFMedium DMF DMF Tween 20 Tween 20 Strain IT C I C I C I C I Listeria 24 hM 2 M 1 M 2 M 2 innocua 36 48 h M 3 M 2 M 3 M 3 Listeria 24 h M 1.5 M0.5 M 1.5 M 1.5 ivanovii 18 48 h M 2.5 M 2 M 2.5 M 2.5 Listeria 24 h M 2M 1 M 2 M 2 monocytogenes 23 48 h M 3 M 2 M 3 M 3 Staphylococcus 24 h —— — — — — — — aureus 20 48 h — — — — — — — — Pseudomonas 24 h — — — — —— — — aeruginosa 165 48 h — — — — — — — —

TABLE 19 stored for stored for fresh 9 weeks fresh 9 weeks DMF DMFMedium DMF DMF Tween 20 Tween 20 Strain IT C I C I C I C I Listeria 24 hM 2 M 0.5 M 2 M 1.5 innocua 36 48 h M 3 M 1 M 3 M 2.5 Listeria 24 h M1.5 M — M 1.5 M 1 ivanovii 18 48 h M 2.5 M 0.5 M 2.5 M 2.5 Listeria 24 hM 2 M 0.5 M 2 M 1 monocytogenes 23 48 h M 3 M 0.5 M 3 M 2 Staphylococcus24 h — — — — — — — — aureus 20 48 h — — — — — — — — Pseudomonas 24 h — —— — — — — — aeruginosa 165 48 h — — — — — — — —

TABLE 20 stored for stored for fresh 12 weeks fresh 12 weeks DMF DMFMedium DMF DMF Tween 20 Tween 20 Strain IT C I C I C I C I Listeria 24 hM 2 M — M 2 M 1 innocua 36 48 h M 3 M 0.5 M 3 M 2.5 Listeria 24 h M 1.5M — M 1.5 M 1 ivanovii 18 48 h M 2.5 M — M 2.5 M 2 Listeria 24 h M 2 M —M 2 M 1 monocytogenes 23 48 h M 3 M — M 3 M 2 Staphylococcus 24 h — — —— — — — — aureus 20 48 h — — — — — — — — Pseudomonas 24 h — — — — — — —— aeruginosa 165 48 h — — — — — — — — I: intensity of coloration(arbitrary scale); C: color; IT: incubation time; M: magenta; —:colorless

The medium containing the substrate dissolved in DMF alone exhibits avery substantial drop in the expression of β-N-acetylglucosaminidaseactivity after storage for three weeks. This drop is amplified overtime. Activity can no longer be detected after twelve weeks. The mediumcontaining the substrate dissolved in a mixture of DMF and Tween 20exhibits a constant expression of β-N-acetylglucosaminidase activityover time up to at least six weeks. After nine and twelve weeks, thereis a slight but not prohibitive drop in the expression ofβ-N-acetylglucosaminidase activity. Tween 20 therefore makes it possibleto stabilize the β-N-acetylglucosaminidase substrate and makes it easierto store and use culture media containing this type of substrate.

In the above Examples, the numbers following the strains correspond tothe number of each strain referenced in the Applicant's collection. Theintensity of coloration corresponds to an arbitrary scale defined asfollows:

0 no activity 0.1 trace of coloration 0.5 very pale coloration 1distinct coloration of low intensity 2 clear coloration of mediumintensity 3 intense coloration 4 very intense coloration

1. A medium for detecting and/or identifying microorganisms with anenzymatic activity selected from the group consisting of esteraseactivity, osidase activity, peptidase activity, sulfatase activity,phosphatase activity, and mixtures thereof, said medium comprising aculture medium and at least one chromogenic or fluorogenic substrateselected from the group consisting of esterase substrate, osidasesubstrate, peptidase substrate, sulfatase substrate, phosphatasesubstrate, and mixtures thereof with the exclusion of substratescomprising a4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)vinyl]quinolinium-1-(propan-3-ylcarboxylicacid) cation and an anion, the detecting and/or identifying being basedon revealing the presence of the enzymatic activity, wherein saidmedium: is in a stable, ready-to-use liquid or gel form; and contains:at least one fatty acid sorbitan ester (FASE) or at least one fatty acid(FA) or an FASE/FA mixture as an emulsifying stabilizer, theconcentration of (FASE) in the medium in % by weight is greater than orequal to 0.5% and less than or equal to 5%, and at least one solvent(S).
 2. The medium according to claim 1, obtained by mixing at least onestock solution of substrate in the solvent S, with FASE, FA or anFASE/FA mixture, and with at least one component of the culture medium.3. The medium according to claim 1, wherein the FASE is selected fromthe group consisting of: polyethoxylated sorbitan monolaurate containing20 units of ethylene oxide (EO); polyethoxylated sorbitan monopalmitate(20 EO); polyethoxylated sorbitan monostearate (20 EO); polyethoxylatedsorbitan tristearate (20 EO); polyethoxylated sorbitan monooleate (20EO); polyethoxylated sorbitan sesquioleate (20 EO); polyethoxylatedsorbitan trioleate (20 EO); and mixtures thereof.
 4. The mediumaccording to claim 1, wherein the FA is selected from the groupconsisting of C4–C20 saturated or unsaturated fatty acids, and mixturesthereof.
 5. The medium according to claim 1, also comprising at leastone anionic surfactant.
 6. The medium according to claim 1, wherein thechromogenic or fluorogenic substrate consists of a target part for theenzyme and a chromophoric or fluorophoric part, the target part beingselected from the group consisting of: glycosides consisting of mono-,di- and/or polysaccharide units linked in a α- or β-position to ahydroxyl group of the fluorophoric or chromophoric part; α-amino acidsor peptides; organic acids such as —O—CO(CH₂)_(n)—CH₃, where n isbetween 0 and 20; and a sulfate, a phosphate, a pyrosulfate, apyrophosphate and a phosphor-diester, and the chromophoric orfluorophoric part being selected from the group consisting of:quinones/anthraquinones and derivatives; amino- and hydroxycoumarins andderivatives; fluoresceins and derivatives; and indoxyls and derivatives.7. The medium according to claim 1, wherein the solvent S is selectedfrom the group consisting of: alcohols; amides; sulfur-containingsolvents; aqueous solvents; and mixtures thereof.
 8. The mediumaccording to claim 1, wherein the proportion by weight of fatty acidsorbitan ester(s) (FASE) to solvent (S) is between 20:80 and 80:20. 9.The medium according to claim 1, wherein the concentration of substratein the medium is defined as follows in mg/l:1≦substrate≦2000.
 10. The medium according to claim 1, wherein theculture medium is selected from the group consisting of selective mediaof the type consisting of MacConkey, Columbia ANC, PALCAM and Sabouraudgentamycin-chloramphenicol media; and non-selective media of the typeconsisting of Columbia ± blood, trypcase soya, nutrient gelose andSabouraud media.
 11. The medium according to claim 1, wherein themicroorganisms are of the genus Salmonella, Pseudomonas, Listeria,Staphylococcus, Enterococcus or Candida.
 12. The medium according toclaim 1, wherein the concentration of (FASE) in the medium in % byweight is greater than or equal to 1.5% and less than or equal to 3.5%.13. A method of obtaining a medium for detecting/identifyingmicro-organisms with an enzymatic activity selected from the groupconsisting of esterase activity, osidase activity, peptidase activity,sulfatase activity, phosphatase activity, and mixtures thereof, saidmedium comprising a culture medium and at least one chromogenic orfluorogenic substrate selected from the group consisting of esterasesubstrate, osidase substrate, peptidase substrate, sulfatase substrate,phosphatase substrate, or mixtures thereof, with the exclusion ofsubstrates comprising a4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)vinyl]quinolinium-1-(propan-3-ylcarboxylicacid) cation and an anion, the detecting and/or identifying being basedon revealing the presence of the enzymatic activity, wherein saidmedium: is in a stable, ready-to-use liquid or gel form; and contains:at least one fatty acid sorbitan ester (FASE) or at least one fatty acid(FA) or an FASE/FA mixture as an emulsifying stabilizer, theconcentration of (FASE) in the medium in % by weight is greater than orequal to 0.5% and less than or equal to 5%, and at least one solvent (S)said method comprising the steps of: preparing at least one stocksolution of the chromogenic or fluorogenic substrate in the solvent Sand of at least one FASE, FA or FASE/FA mixture, adding the stocksolution to the culture medium, and homogenizing the stock solution andthe culture medium.
 14. The method according to claim 13, wherein theconcentration of (FASE) in the medium in % by weight is greater than orequal to 1.5% and less than or equal to 3.5%.
 15. A method fordetection/identification of microorganisms with an enzymatic activityselected from the group consisting of esterase activity, osidaseactivity, peptidase activity, sulfatase activity, phosphatase activity,or mixtures thereof the method comprising stabilizing a medium (liquidor gel) with a fatty acid sorbitan ester (FASE), fatty acid (FA) orFASE/FA as an emulsifying stabilizer, the FASE having a concentration inthe medium in % by weight of greater than or equal to 0.5% and less thanor equal to 5%, the medium comprising a culture medium and at least onechromogenic or fluorogenic substrate selected from the group consistingof esterase substrate, osidase substrate, peptidase substrate, sulfatasesubstrate, phosphatase substrate, and mixtures thereof with theexclusion of substrates comprising a4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)vinyl]quinolinium-1-(propan-3-ylcarboxylicacid) cation and an anion, in association with at least one solvent Sfor the chromogenic or fluorogenic substrate present in the medium;inoculating the culture medium with microorganisms to bedetected/identified; incubating the inoculated medium under appropriateconditions; determining colorations around colonies in the medium; andcorrelating the colorations around the colonies to reveal hydrolysis ofthe chromogenic or fluorogenic substrate by the microorganisms anddetect/identify the microorganisms.
 16. The method according to claim15, wherein the concentration of (FASE) in the medium in % by weight isgreater than or equal to 1.5% and less than or equal to 3.5%.